The present invention provides polyacrylamide hydrogels (particularly polyacrylamide hydrogel arrays) that are made from prepolymers (including especially polyacrylamide reactive polymers), as well as compositions and novel methods for their preparation. In particular, the various elements of the invention allow preparation of a polyacrylamide hydrogel having a specific pore size (and a more uniform hydrogel pore size) that is appropriate, for instance, for DNA applications, and further desirably is non-opaque. Additionally, the polyacrylamide reactive prepolymer used for crosslinking to obtain the polyacrylamide hydrogel is of sufficient viscosity that it can be employed in a commercial coating process.
Acrylamide (CH2xe2x95x90CHCONH2; C.A.S. 79-06-1; also known as acrylamide monomer, acrylic amide, propenamide, and 2-propenamide) is an odorless, free-flowing white crystalline substance that is used as a chemical intermediate in the production and synthesis of polyacrylamides. These high molecular weight polymers have a variety of uses and further can be modified to optimize nonionic, anionic, or cationic properties for specified uses.
Polyacrylamide hydrogels are especially employed as molecular sieves for the separation of nucleic acids, proteins, and other moieties, and as binding layers to adhere to surfaces biological molecules including, but not limited to, proteins, peptides, oligonucleotides, polynucleotides, and larger nucleic acid fragments. The gels currently are produced as thin sheets or slabs, typically by depositing a solution of acrylamide monomer, a crosslinker such methylene bisacrylamide, and an initiator such as N,N,Nxe2x80x2,Nxe2x80x2-tetramethylethylendiamine (TEMED) in between two glass surfaces (e.g., glass plates or microscope slides) using a spacer to obtain the desired thickness of polyacrylamide. Generally, the acrylamide polymerization solution is a 4-5% solution (acrylamide/bisacrylamide 19/1) in water/glycerol, with a nominal amount of initiator added. The solution is polymerized and crosslinked either by ultraviolet (UV) radiation (e.g., 254 nm for at least about 15 minutes, or other appropriate UV conditions, collectively termed xe2x80x9cphotopolymerizationxe2x80x9d), or by thermal initiation at elevated temperature (e.g., typically at about 40xc2x0 C.). Following polymerization and crosslinking, the top glass slide is removed from the surface to uncover the gel. The pore size (or xe2x80x9csieving propertiesxe2x80x9d) of the gel is controlled by changing the amount of crosslinker and the % solids in the monomer solution. The pore size also can be controlled by changing the polymerization temperature.
In the fabrication of polyacrylamide hydrogel arrays (i.e., patterned gels) used as binding layers for biological molecules, the acrylamide solution typically is imaged through a mask during the UV polymerization/crosslinking step. The top glass slide is removed after polymerization, and the unpolymerized monomer is washed away (developed) with water leaving a fine feature pattern of polyacrylamide hydrogel, the crosslinked polyacrylamide hydrogel pads. Further, in an application of lithographic techniques known in the semiconductor industry, light can be applied to discrete locations on the surface of a polyacrylamide hydrogel to activate these specified regions for the attachment of an anti-ligand, such as an antibody or antigen, hormone or hormone receptor, oligonucleotide, or polysaccharide on the surface (e.g., a polyacrylamide hydrogel surface) of a solid support (PCT International Application WO 91/07087, incorporated by reference).
The current approach of making polyacrylamide hydrogels starting from acrylamide monomer has several disadvantages, some of which are described below:
1. The coating process is difficult and expensive to automate because the film thickness is controlled with a spacer and top glass plate. The removal of the top glass plate must be done manually. Due to difficulties automating the process (e.g., viscosity too low for commercial coating methods), the coating of the monomer solution currently is done manually.
2. The reaction time of the acrylamide is excessively long (e.g., typically from about 15 to about 90 minutes at a short wavelength of about 254 nm), making the UV polymerization and crosslinking step incompatible with standard imaging equipment such as mask aligners and photoprinters.
3. The acrylamide in monomer form is a neurotoxin and a carcinogen which makes coating, handling, and waste disposal of the material hazardous and expensive.
4. The polymerization process is inherently difficult to control, and frequently results in random and non-uniform pore size and crosslink density. In particular, using current approaches, it is difficult to obtain a sufficiently rigid polyacrylamide hydrogel having a large enough pore size such as can be employed for use with applications involving DNA or DNA fragments.
Added to these disadvantages are the further problems that crystallization of monomer frequently occurs on commonly used equipment and laboratory surfaces, and exothermic polymerization can occur in coating reservoirs, necessitating the use of stabilizers (which also are known as inhibitors).
The present invention seeks to overcome some of the aforesaid disadvantages of the prior art. In particular, the present invention provides polyacrylamide hydrogels (particularly hydrogel arrays) made from prepolymers (and particularly, polyacrylamide reactive polymers), novel methods of preparation, and prepolymer compositions, which avoid these difficulties attendant the prior art. The present invention can be employed in an economic fashion in commercial processes which require polyacrylamide hydrogels/hydrogel pads, thus reducing manufacturing times and enhancing throughput. The present invention further provides improved control of crosslink density, resulting in a more uniform hydrogel pore size, and production of a polyacrylamide hydrogel appropriate for use with DNA. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention relates to methods for preparing a polyacrylamide hydrogel or hydrogel array comprising: (a) preparing a polyacrylamide reactive prepolymer having a controlled size; and (b) crosslinking the polyacrylamide reactive prepolymer to obtain the polyacrylamide hydrogel or hydrogel array. Making a polyacrylamide hydrogel array preferably comprises additional steps, optionally, developing the pattern in the array, and further optimally selectively removing the uncrosslinked polymer in aqueous solution to produce the hydrogel array. Pattern development desirably is accomplished by exposing the polyacrylamide reactive prepolymer through a photomask. The present invention further provides compositions and novel methods for preparing the polyacrylamide reactive prepolymer. Thus, the invention allows preparation of a specific pore size (and more uniforn pore size) polyacrylamide hydrogel which can be employed, inter alia, in applications involving DNA, and optimally, is non-opaque. Furthermore, the methods of the invention are advantageous in that the polyacrylamide reactive prepolymer used for crosslinking to obtain the polyacrylamide hydrogel is of sufficient viscosity that it can be employed in a commercial coating process.